Aquaspillium arcticum is a psychrophilic bacterium that was isolated from arctic sediment and grows optimally at 4°C. We have cloned, purified, and characterized malate dehydrogenase from A. arcticum (Aa MDH). We also have determined the crystal structures of apo-Aa MDH, Aa MDH⅐NADH binary complex, and Aa MDH⅐NAD⅐oxaloacetate ternary complex at 1.9-, 2.1-, and 2.5-Å resolutions, respectively. The Aa MDH sequence is most closely related to the sequence of a thermophilic MDH from Thermus flavus (Tf MDH), showing 61% sequence identity and over 90% sequence similarity. Stability studies show that Aa MDH has a half-life of 10 min at 55°C, whereas Tf MDH is fully active at 90°C for 1 h. Aa MDH shows 2-3-fold higher catalytic efficiency compared with a mesophilic or a thermophilic MDH at the temperature range 4 -10°C. Structural comparison of Aa MDH and Tf MDH suggests that the increased relative flexibility of active site residues, favorable surface charge distribution for substrate and cofactor, and the reduced intersubunit ion pair interactions may be the major factors for the efficient catalytic activity of Aa MDH at low temperatures.Psychrophiles grow at low temperatures, where most of other organisms cannot grow. In order to survive such extreme environments (less than 4°C), enzymes from psychrophiles must catalyze efficiently at low temperatures (1-6). While good progress is being made to elucidate the adaptation mechanism of enzymes from some extremophiles including hyperthermophile, the molecular basis of cold adaptation of psychrophilic enzymes is relatively poorly understood (7-9). However, psychrophilic enzymes have generated considerable interest, since they can be used to improve the efficiency of industrial processes and for environmental applications (1, 10). Also, comparison of the structures of psychrophilic enzymes with mesophilic, thermophilic, and hyperthermophilic counterparts may add new insights into the understanding of catalytic mechanism and analysis of thermostability factors.As a first step to understand the structural basis of cold adaptation of psychrophilic enzymes, we have carried out biochemical and structural studies of malate dehydrogenase from Aquaspillium arcticum, a psychrophilic bacterium that was isolated from Arctic sediments and grows optimally at 4°C (11).MDH 1 is a homodimeric enzyme that catalyzes the reversible oxidation of malate to oxaloacetate in the presence of NAD in the citric acid cycle and thus plays a major role in central metabolism (12). Therefore, a certain amount of MDH is always expected to be present in most living organisms. Several MDHs from different sources have been extensively studied in genetic and biochemical aspects; sequences of a large number of malate dehydrogenases from organisms representing Archea, Bacteria, and Eukarya have been reported, and many of their gene products have been characterized. Furthermore, crystal structures of MDHs from the thermophile Thermus flavus (13), the mesophile Escherichia coli (14, 15), porcine heart mytochondria ...

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